Blackwell Publishing LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066The Linnean Society of London, 2006?

2006
892
197211
Original Article
GENETIC AND MORPHOLOGICAL DIFFERENTIATION IN DUNG BEETLE SISTER SPECIES
A. PIZZO
ET AL

Biological Journal of the Linnean Society, 2006, 89, 197211. With 8 figures

Genetic and morphological differentiation patterns

between sister species: the case of Onthophagus taurus
and Onthophagus illyricus (Coleoptera, Scarabaeidae)
ASTRID PIZZO*, ANGELA ROGGERO, CLAUDIA PALESTRINI, PIERO CERVELLA,
MASSIMILIANO DEL PERO and ANTONIO ROLANDO
Dipartimento di Biologia Animale e dellUomo, Universit degli Studi di Torino, via Accademia Albertina
13, I-10123 Torino, Italy
Received 5 December 2004; accepted for publication 1 December 2005

The present study investigated the morphological and genetic differentiation pattern between two sympatric dung
beetle sister species, Onthophagus taurus and Onthophagus illyricus. The geometric morphometric approach coupled
with the use of molecular markers allowed examination of the nature of interspecific relationships and an outline of
the evolutionary and geographical processes that might have led to interspecific differentiation and the present-day
partial sympatric and syntopic pattern of distribution. Geometric morphometrics failed to discrimininate the two
species on the ground of external morphological traits, but revealed interspecific differences when the shape of the
primary sexual traits was taken into account. The use of two different molecular markers (cytochrome oxidase subunit I gene and amplified fragment-length polymorphism) demonstrates that the two species are genetically well differentiated, forming two distinguishable lineages probably diverged during the Pliocene by allopatric speciation. No
evidence of past or recent introgression and no support for hybridization were found, suggesting that sympatry was
established subsequently, when speciation was accomplished. Both molecular markers and genital shape indicate
that phenotypically intermediate individuals, despite their ambiguous appearance, are members of O. illyricus species rather than hybrids. 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006,
89, 197211.

ADDITIONAL KEYWORDS: AFLP allopatric speciation COI gene external morphology genital
morphology geometric morphometrics interspecific shape variation mtDNA.

INTRODUCTION
Sister species, by definition, are derived from a common ancestor, shared by no other species and therefore
they are important models to study evolutionary
events that promote speciation. The importance of
identifying patterns of differentiation between closelyrelated species, was already recognized by Darwin
(1859) as a basis for understanding evolution by natural selection. Sister species often occur in sympatry
(Barraclough & Vogler, 2000), or share portions of the
distribution areas sometimes representing hybrid
zones, which provide an opportunity to investigate
genetic and ecological interactions between species
(Hardig et al., 2000). It has been suggested that sym.
*Corresponding author. E-mail: astrid.pizzo@unito.it

patric species which are also syntopic may have experienced the same environmental influences, at least
throughout their most recent evolutionary history
(Dawson et al., 2002).
The morphological similarity of sister species and
the existence of forms with intermediate morphological traits, together with their occurrence in sympatry
and in syntopy, have been interpreted in several ways.
Some authors consider that these conditions hint at
the possibility that speciation occurred in sympatry
(Seehausen, Witte & van Alphen; Barraclough &
Vogler, 2000; Barluenga & Meyer, 2004). Among the
several theories of speciation, sympatric speciation
has been the most controversial, but a growing body of
empirical data shows that a variety of evolutionary
processes can result in differentiation under sympatric conditions: the main accepted cause of divergence

2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211

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A. PIZZO ET AL.

in sympatric populations is the ecological specialization to local environments (Kondrashov, Yampolsky &
Shabalina, 1998; Schluter, 2000), but sympatric
divergence trends on a large spatial scale have also
been hypothesized (Salewski, 2003). Sexual selection
through assortative mating (Seehausen et al., 1998;
Gavrilets & Waxman, 2002) and sexual conflict
(Arnqvist et al., 2000) have also been proposed as
mechanisms possibly promoting divergence between
sympatric populations.
However, it is believed that most species evolved in
allopatry by divergence of geographically-isolated populations from ancestral species (Mayr, 1963). Allopatric speciation, due to barriers that hinder gene flow,
may be followed by subsequent re-colonizations and
re-establishment of sympatry by the sister species
(Bernatchez & Dodson, 1990). In these cases, when
interspecific barriers are not completely developed,
hybridization can occur in the overlap zone.
Speciation has long been thought to involve a process of genetic divergence between populations coupled with a secondary acquisition of morphological
differences (Mayr, 1963). Nevertheless, species deriving from a common ancestor may show genetical
divergence without accompanying morphological
changes (Mathews et al., 2002). In these cases, the correct classification and interpretation of interspecific
relationships could be obtained only when genetic
data are considered (Parson & Shaw, 2001).
Onthophagus taurus Schreber 1759 and Onthophagus illyricus Scopoli 1763 are the only two European
species of the subgenus Onthophagus s.s. (Zunino,
1979), usually considered sister species for their
noticeable morphological similarity (Balthasar, 1963;
Baraud, 1992; Lohse & Lucht, 1992; Moczek & Emlen,
1999; Martin-Piera & Lopez-Colon, 2000). Onthophagus taurus shows a typical TuranicEuropean
Mediterranean distribution (Balthasar, 1963). The
chorology of O. illyricus is TuranicEuropean, and its
distribution greatly overlaps with that of O. taurus
but, because of the unreliability of some geographical
data, its actual distribution is still imprecise (MartinPiera & Lopez-Colon, 2000). However, in the extensive
overlap zone, the two species often occur in syntopy.
Males of both species exhibit alternative phenotypes
(male polyphenism); individuals larger than a critical
body size threshold develop a pair of disproportionated
head horns (major or horned males), whereas
smaller minor males develop only rudimental horns
or remain hornless (Moczek, 1996, 1998; Emlen &
Nijhout, 1999; Moczek & Emlen, 1999; Simmons,
Tomkins & Hunt, 1999; Hunt & Simmons, 2000;
Moczek & Emlen, 2000; Palestrini, Rolando & Laiolo,
2000; Hunt & Simmons, 2002).
According to the traditional classification criteria
(Janssens, 1960; Paulian & Baraud, 1982), differences

between O. taurus and O. illyricus only concern the

surface of the elytra. Onthophagus illyricus elytra
have an evident granulation on the sutural intervals
(interstriae) and are pubescent, entirely covered with
short setae, whereas those of O. taurus are smooth,
with a light punctation on the sutural intervals and
without setae in the central disc. All other external
characters are considered to be identical in the two
species.
Although most individuals are easily identified following these criteria, some others are not because of
intermediate pubescence and granulation of the elytra
(Paulian & Baraud, 1982). In these instances, the
shape of the reproductive system often provides the
only reliable character for species identification
(Zunino, 1971; Eberhard, 1985). As reported by
Howden & Gill (1993), the members of the genus
Onthophagus show rapid and divergent variation in
genital morphology as result of intense sexual selection (Eberhard, 1985), and therefore reproductive
traits are diagnostic for species identification. However, Paulian & Baraud (1982) pointed out that intermediate pubescence of the elytra was related to
intermediate genital features.
The present study aimed to analyse the morphological and genetic differentiation patterns between
closely-related species, using the sister species pair
O. taurus and O. illyricus as a model.
The comparative analyses of morphological traits
and genetic diversity of this species pair led the
present study to: (1) investigate the historical and
recent differentiation patterns of these sister species
at different levels; (2) determine the actual taxonomic
status of individuals with intermediate phenotypes
and, concurrently, establish whether hybridization
events occurred; and (3) hypothesize timing and mechanisms involved in the differentiation processes.

MATERIAL AND METHODS

GEOMETRIC

MORPHOMETRIC ANALYSIS

The geometric morphometric technique (Bookstein,

1991; Rohlf & Marcus, 1993; Marcus et al., 1996;
Adams, Slice & Rohlf, 2004) is more powerful to
describe shape variation than the traditional morphometrics and has proved to be an useful tool for solving
a variety of biological problems. Here, the landmark
geometric technique was used to quantify and analyse
the interspecific differentiation in the shape of three
external (i.e. head, pronotum and elytra) and two genital (vagina and aedeagus) morphological structures
of 180 females and 157 males of O. taurus and
O. illyricus collected in 2003 in La Mandria Natural
Park (North-western Italy), where individuals with
intermediate phenotypic condition (hereafter called

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199

Table 1. The sample considered in this study

N (geometric
morphometrics)

N (CO I)

N (AFLP)

Species

Sampling locality

Region, country

Females

Males

Females

Females

Onthophagus taurus

La Mandria
Brlog
Aosta
Modena
Caserta
Toulon
Doana
La Mandria
Brlog
Trieste
Caserta
La Mandria

Piemonte, Italy
Croatia
Western Alps, Italy
Emilia, Italy
Campania, Italy
France
Spain
Piemonte, Italy
Croatia
Friuli, Italy
Campania-Italy
Piemonte-Italy

79

81

20

70

70

17

5
2
2
2
2
2
2
5
2
3
2
4

9
7

5
9

Onthophagus illyricus

IUTS

For each species, sampling locality, region-country and number of individuals used in each analysis are given. COI,
cytochrome oxidase subunit I; AFLP, amplified fragment-length polymorphism; IUTS, individuals with intermediate
phenotypic condition.

Figure 1. Drawings of head of both sexes, pronotum, and elytra of Onthophagus taurus and Onthophagus illyricus.
Photographs of pronota and elytra were taken at 12.5 magnification, whereas heads are at 32 magnification The
landmarks for head (N = 5), pronotum (N = 4), and elytra (N = 7) were digitized on half of each structure to remove the
variability introduced by an eventual asymmetry.

IUTS) were found (Table 1). Images of each anatomical structure were captured using a digital camera
Olympus DP11 connected to a stereoscopic microscope
Leica MZ8, taking care to align the edges of each
structure on the same horizontal plane. Genitalia

were photographed on a thin film of glycerol to avoid

deformation of parts due to compression under the
cover slides. Landmarks were digitized using TpsDig
1.37 (Rohlf, 2003a) and were positioned as shown in
Figure 1 (external morphology), Figure 2 (vagina), and

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A. PIZZO ET AL.

Figure 2. Female genitalia: the sclerotized area of the vagina and the landmarks ( N = 5) used in the geometric morphometric analysis. Genitalia were held with a pair of tweezers in a thin film of glycerol and photographed at 50 magnification. The two most extremely different morphologies are shown.

Figure 3. Male genitalia: the aedeagus and the position of

the landmarks (N = 9) on the side of the left paramer is
revealed. The two most extremely different morphologies
are shown. Genitalia were held with a tweezers in a thin
film of glycerol and photographed at 50 magnification.

Figure 3 (left paramere of aedeagus). They were chosen for their relative ease of identification, their
homology in the two species, and the ability of the
suite of landmarks to capture the general shape of
each morphological structure.
To evaluate the confidence of the landmark configuration, a repeatability test was conducted by digitizing the landmarks ten times on the same specimen
and then calculating the ratio between the variance
on the same specimen and the variance of the total
sample (variance = (Procrustes distances) 2/n 1,
where n is the number of objects considered in each
set of measures). The landmark configuration was
accepted only if the ratio was less than or equal to
0.05.

The thin-plane spline (TPS) method (Bookstein,

1989) was used to visualize a tangent space for the statistical analysis of shape variation. Generalized procrustean superimposition (Rohlf, 1990; Rohlf & Slice,
1990), calculation of the centroid size, partial warps,
relative warps and visualization of deformation grids,
which allowed a description of shape variation, were
performed using tpsRelw 1.33 (Rohlf, 2003b). Grouping in classes and visualizations of the samples on an
axis system were made with the software NTSYSpc
2.11 (Rohlf, 19982002) using the two first relative
warp scores.
Discriminant analysis was conducted on the relative
warp scores of each considered shape to obtain a classification matrix based on the shape variation. The
percentages of correct classification were used to evaluate the interspecific discriminatory power of each
anatomical structure. Initially, three groups (i.e.
O. taurus, O. illyricus and IUTS) were considered and
then incorporated IUTS in the group suggested by the
relative warp plot. To determine whether a certain
amount of intraspecific shape variation was related to
differences in size of each structure (Rosenberg, 2001),
the values of the centroid size and the values of the
first relative warp of head, pronotum, elytra and genitalia in each sex and species were correlated. Discriminant analysis and correlation were computed
using the package SYSTAT 8.0 (Wilkinson, 1998).

MOLECULAR

MARKERS ANALYSES

Mitochondrial and nuclear markers can provide fine

details on the degree of genetic differentiation
resulted from historical and recent processes,
respectively. We also employed the amplified fragment-length polymorphism (AFLP) technique, which
has been used successfully in several animal taxa at
different taxonomical levels (Vos et al., 1995; Liu et al.,
1998; Albertson et al., 1999; Liu et al., 1999; De Knijff
et al., 2001; Giannasi, Thorpe & Malhotra, 2001;

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GENETIC AND MORPHOLOGICAL DIFFERENTIATION IN DUNG BEETLE SISTER SPECIES

Ogden & Thorpe, 2002), but has been employed in
insects only recently (Parsons & Shaw, 2001; Carisio
et al., 2004) to evaluate the recent genetic
differentiation at the nuclear genome level. Recent
hybridization occurrences were investigated through
AFLP markers, which allow the simultaneous analyses of several loci, thus increasing the possibility of
hybrid identification (Congiu et al., 2001).
Analyses were conducted on individuals collected in
Italy, Croatia, France, and Spain during the year 2003
and then preserved in 100% ethanol. After rehydratation in distilled water, total DNA was extracted from
the heads by proteinase K/SDS, overnight digestion,
chloroform extraction and ethanol precipitation
according to standard protocols (Laird et al., 1991).
mtDNA: polymerase chain reaction (PCR)
amplification, sequencing and analysis
The sample was composed of 17 O. taurus, 12
O. illyricus and four IUTS randomly selected from the
female sample used for geometric mophometric analyses, as indicated in Table 1. A 597-bp fragment of
the cytochrome oxidase subunit I (COI) gene was
amplified by PCR using primers C1-J-2441 (5 -CCA
ACAGGAATTAAAATTTTTAGATGATTAGC-3) and
TL2-N-3014 (5-TCCAATGCACTAGCCAGAATCTGC
CATATTA-3) (Simon et al., 1994). Amplifications
were performed in a volume of 25 L in 10 mM TrisHCl, 50 mM KCl, 0.1% Triton, 4.0 mM MgCl2, 0.1 mM
each dNTP, 0.5 M each primer and Taq DNA polymerase (0.5 U, Fischer). The amplification was carried
out in an iCycler Biorad thermocycler in the following
conditions: 95 C initial denaturation for 5 min, 40
cycles of 94 C for 60 s, 50 C for 60 s, 72 C for 90 s,
and a final elongation at 72 C for 10 min. PCR products were purified and directly sequenced using a
Thermo Sequenase Cy5 Dye Terminator Kit (Amersham Pharmacia Biotech) following the manufacturers instructions. All sequencing was performed in
both directions and the haplotype of each individual
was verified on the basis of both sequences.
The average nucleotide diversity within each
species S and the net nucleotide diversity between species dA (Nei, 1987) was estimated as:
dA = dXY [(dX + dY)/2], where dXY is the nucleotide
divergence between species X and Y, and dX and dY are
the average interspecific nucleotide differences.
Time of divergence of species were estimated following the equation dA = 2T (Nei, 1987) assuming the
standard mutation rate for arthropod mtDNA of 1.2%
per million years for (Brower, 1994).
For phylogenetic analyses, both distance and parsimony methods were used. A distance-based tree was
generated using the MEGA 2.1 software (Kumar
et al., 2001). Sequences obtained were analysed
toghether with those deposited in the GenBank

201

(Villalba et al., 2002) referring to some European species of the genus Onthophagus and other two species
(Euoniticellus pallipes and Euoniticellus fulvus) used
as an outgroup.
Pairwise distances between haplotypes were
obtained under the assumption of the JukesCantor
model, and the tree was constructed by the Neighbourjoining (NJ) method (Saitou & Nei, 1987). Robustness
of the inferred trees was tested by bootstrapping
(Felsenstein, 1985) with 1000 replications. An
unweighted maximum parsimony (MP) analysis
(PAUP, version 4.0, Beta 10, Swofford, 1990) was performed using a heuristic search with 100 random
sequence additions and TBR branch swapping.
AFLP markers
The AFLP method was performed as described by Vos
et al. (1995) with a few modifications. Genomic DNA
was cut using the restriction enzymes EcoRI and MseI,
and double-stranded EcoRI and MseI adapters were
ligated to the sticky ends of the fragments. Preselective PCR was carried out with 1 : 5 template dilutions
and with an EcoRI primer (EcoRI adaptor sequence)
and MseI primer (MseI adaptor sequence + 1 nucleotide). Dilutions (1 : 20) of the preselective PCR
products were used as templates for selective amplification, which was performed with an EcoRI primer
containing two selective nucleotides and a MseI
primer containing three selective nucleotides. An initial screening using six selective primer combinations
was performed on 12 individuals (three O. taurus and
three O. illyricus from La Mandria and as many from
Brlog). The primer combinations giving clear, reproducible, and polymorphic electrophoretic patterns
were chosen for further studies. Analyses were performed on a total of 34 individuals, as indicated in
Table 1. The AFLP adapter sequences, preamplification primer sequences, and selective amplification
primer sequences were as previously reported by Carisio et al. (2004).
Selective amplification products were separated
with an AFLexpressII automated sequencer (AP Biotech). Fragments were automatically scored by the
program ALFwin Fragment Analyser 1.0 (AP Biotech).
When peak height exceeded the standard parametersetting threshold, a peak (i.e. a fragment) was scored
as present (1), otherwise it was scored as absent (0).
An additional visual check of gels was made to correct
possible misinterpretations of automated procedures.
For each combination, all loci showing a clear and
unambiguous banding pattern were scored, whereas
uncertain peaks were considered as missing data.
Results of scoring were exported to a presence/absence
matrix and used for further analyses. Band sizes were
estimated using a standard size marker (Gitelman &
Davis, 1997).

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A. PIZZO ET AL.

Data obtained from each primer pair were combined

and analysed as a single matrix. From AFLP presence/
absence data, a dissimilarity matrix was generated
using Gowers general similarity coefficient (Sneath &
Sokal, 1973) and a principal coordinate analysis
(PCoA) was performed on the dissimilarity matrix.
Resulting principal coordinate scores were then plotted to evaluate the usefulness of AFLP in discriminating between species.
Genetic diversity and population genetic structure
analyses were performed using the program AFLPSURV (Vekemans, 2002). AFLPs are assumed to generate dominant markers and heterozygotes cannot be
distinguished directly. Estimates of allele frequencies
of AFLP loci were calculated according to the method
described in Zhivotovsky (1999). This approach
assumes that populations are in HardyWeinberg
equilibrium and that AFLPs produce two alleles per
locus. At a particular locus, the plus-allele (the presence of a band) dominates over the null-allele (unamplifiable by PCR). The frequency of the null allele at
each locus is calculated by means of a Bayesian
method from the sample size and the number of individuals lacking the AFLP fragment. The following
parameters were then calculated for each population:
the proportion of polymorphic loci (PPL) at the 5%
level and the expected heterozygosity or Neis gene
diversity Hj (Lynch & Milligan, 1994). The parameters
of population and species genetic structure were: the
total gene diversity HT, the average gene diversity
within populations HW, analogous to Neis HS (Nei,
1987), the average gene diversity among populations
in excess of that observed within populations HB, analogous to Neis DST (Nei, 1987), and Wrights fixation
index FST (Wright, 1969). The significance of FST was
tested with 1000 random permutations. Pairwise
genetic distances between populations and species
were estimated with Neis D (Lynch & Milligan, 1994).
These analyses were performed comparing first the
two species independently from the geographical origin and then the two species in each locality. Finally,
the parameters of intraspecific differences were measured. The matrix of pairwise genetic distances
between each pairs of individuals in the overall
dataset was used to build a tree of individuals with the
software package PHYLIP, version 3.57c (Felsenstein,
1993) using the NJ procedure.

RESULTS
GEOMETRIC

MORPHOMETRIC ANALYSIS

Values of the first two relative warp scores obtained

from TPS analysis of each anatomical structure were
plotted on an axis system. The contemporary survey of
these relative warp plots and the visual inspection of

deformation grids may lead to some generalizations

about trends of shape variation. As a rule, plots
concerning pronotum, head, and elytra described
intraspecific shape variations but did not allow any
interspecific discrimination (Fig. 4A), except for the

Figure 4. Scatterplots of the two first relative warps

scores obtained from the relative warp analysis of the head
shape of both sexes. Black squares, Onthophagus taurus;
white squares, Onthophagus illyricus; grey squares, IUTS.
A. male head: this plot is presented as an example of the
relative warp plot resulting from the analysis of the shape
of external morphological structures, which does not permit any interspecific discrimination. B, female head: a polygon was drawn encircling the outer individuals of both
species and IUTS. The first axis describes the variation in
the relative positions of anterior margin, vertex carina, and
apex of the neck and the distance between eye and the
genal suture. The dots on the right side of the plot correspond to heads with vertex carina closer to anterior edge
and to the fore of the eye. The second relative warp mainly
refers to the variation in the wideness of the gena; the dots
on the bottom of the plot correspond to sharper and triangular heads whereas those in the upper part of the figure
correspond to more rounded heads.

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GENETIC AND MORPHOLOGICAL DIFFERENTIATION IN DUNG BEETLE SISTER SPECIES

female head plot in which a moderate interspecific
discrimination might be appreciated. IUTS were
encompassed in the polygon outlining O. illyricus individuals (Fig. 4B). Visual inspection of the deformation
grid also revealed that heads of O. taurus females
were more compressed in the fore-hind direction, a little enlarged at the genal level and more triangular
than those of O. illyricus females, most of which were
more elongated and elliptical.
Correlations between centroid size and the scores of
the first relative warp concerning male heads of both
species were significant (r = 0.551, P < 0.0001 for
O. taurus and r = 0.742, P < 0.0001 for O. illyricus),
indicating that a certain amount of shape variation in
this structure was due to the size variation or allometry, probably depending on polyphenism. Female genitalia also showed a moderate significant correlation
between shape and size in both species (r = 0.497,
P < 0.0001 for O. taurus and r = 0.522, P < 0.0001 for
O. illyricus). None of the other anatomical structures
showed linear correlation between shape and size,
suggesting that in these cases shape variation did not
depend on size differences.
Discriminant analysis conducted on relative warp
scores of each structure confirmed that only the
female head allowed some discrimination between
species with 67% O. taurus and 65% O. illyricus correctly classified. However, when IUTS were included
in the O. illyricus group, as suggested by the plot
in Figure 4B, percentages of correct classification
increased, especially for O. illyricus (up to 73% for
O. taurus and 81% for O. illyricus).
All the presented analyses were conducted taking
into account major and minor males separately,
before combining all males together, in the hypothesis that polyphenic male morphology could affect
the results (not shown). These results were not different from those obtained combining male morphs
together.
The shape of female genitalia showed a high interspecific discriminatory power. In the relative warp plot
(Fig. 5A), O. taurus and O. illyricus formed two distinguishable groups, even though a small level of overlap
was visible. Again, all IUTS were incorporated in the
O. illyricus group. Similar results were also obtained
for male genitalia, even though the two species overlapped to a higher degree in this case (Fig. 5B).
Discriminant analysis performed on the relative
warp scores of the female and male genitalia produced
a classification matrix in which only individuals of
O. taurus scored high percentages of correct classification (95% for females and 91% for males) (Table 2).
When IUTS were included in the O. illyricus group,
percentages of correct classifications for O. illyricus
conspicuously increased, whereas those of O. taurus
did not (Table 2).

203

Figure 5. Scatterplots of the two first relative warps

obtained from the relative warp analysis of the genitalia
shape. Black symbols, Onthophagus taurus; white symbols,
Onthophagus illyricus; grey symbols, IUTS.

GENETIC

ANALYSES

Sequence analysis
Alignments of the 597-bp segments of the mitochondrial CO I gene obtained from 33 individuals (and one
O. taurus COI sequence taken from the GenBank)
were straightforward and all sequences were translated into amino acids, suggesting that they are functional. No indels or premature codons were found.
Onthophagus illyricus populations shared a single
haplotype, with a nucleotide composition of A = 30.8%,
C = 15.6%, G = 13% and T = 40.6%, whereas the alignment of O. taurus sequences yielded five different haplotypes with eight polymorphic nucleotide sites and a
mean base composition of A = 31.4%, C = 15.3%,
G = 12.6% and T = 40.7%, which is very similar to that
of O. illyricus.

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Table 2. Results of discriminant analysis on relative warps score of female and male genitalia
Classification after discriminant analysis

Female

II
Male

II

Onthophagus
Onthophagus
IUTS
Onthophagus
Onthophagus
Onthophagus
Onthophagus
IUTS
Onthophagus
Onthophagus

taurus
illyricus
taurus
illyricus
taurus
illyricus
taurus
illyricus

Onthophagus taurus

Onthophagus illyricus

IUTS

% correct

79
81
20
79
101
70
70
17
70
87

75
3
1
75
4
64
1
3
64
6

1
48
10
4
97
2
51
4
6
81

3
30
9

4
18
10

95
59
45
95
96
91
73
59
91
93

I: the three groups [O. taurus, O. illyricus and IUTS, individuals with intermediate phenotypic condition (IUTS)] are
analysed separately. II: the IUTS are included in the O. illyricus group, as suggested by the relative warp plot.

Maximum-parsimony analysis generated four

equally parsimonious trees of 695 steps. The phylogenetic tree resulting from NJ reconstruction revealed
the same basic topology, always showing O. taurus and
O. illyricus as a sister group with high bootstrap values (Fig. 6). The monophyly of each of the two species
was also strongly supported (100%), with individuals
of different species being consistently placed in separate lineages. This preliminary intraspecific survey
showed that the O. taurus clade did not support any
clear geographical subdivision other than the basal
divergence of the haplotype obtained from the Doana
sample.
Mean nucleotide diversity within species was
S = 0.003 for O. taurus and, obviously, S = 0.000 for
O. illyricus. The net nucleotide diversity between the
two species was dA = 0.084 0.013. Using the standard mutation rate for arthropod mtDNA of 1.2% per
million years (Brower, 1994), these data suggest that
the divergence time between the two clades could be
estimated as occurring 34 Mya.
AFLP analysis
After pooling the data from the three AFLP primer
combinations, a total of 96 bands in O. taurus and
87 bands in O. illyricus were scored, ranging from
31350 bp. In O. taurus, 96 bands were found in La
Mandria and 99 in the Brlog population whereas, in
O. illyricus, 80 bands were scored in La Mandria and
84 in Brlog. The percentage of polymorphic bands
(PPL) was slightly lower in O. illyricus (60.4%) than in
O. taurus (66.7%).
The results of PCoA showed that the three primer
combinations produced a very good discrimination
between the two species along the first axis (Fig. 7).
The level of genetic diversity represented by the

average heterozygosity Hj was higher in O. taurus

(0.228) than in O. illyricus (0.184) and, for both species, in Brlog populations (0.249 in O. taurus and
0.193 in O. illyricus) than in La Mandria populations (0.224 in O. taurus and 0.170 in O. illyricus).
The summary statistics of population structure are
presented in Table 3. The total gene diversity HT was
higher in O. taurus than in O. illyricus. Average gene
diversity within populations suggested that most of
the diversity corresponded to within population variation, whereas variation among populations (HB) was
low in both species. Wrights fixation index FST values were 0.027 in O. taurus and 0.075 in O. illyricus.
These results also suggested a low level of differentiation between populations in both species. Statistics
for genetic differentiation among species are presented in Table 3. In the whole sample, the FST value
between species, independent of the origin of
samples, was 0.504, indicating a marked level of
interspecific differentiation. The FST value between
species in each sampled site showed that interspecific differences are comparable in each geographical
locality.
Pairwise Neis genetic distance D between species
was 0.3078. The value of D between species was
slightly higher in Brlog (0.330) than in the La Mandria population (0.307). The value of D between the
two O. taurus populations was 0.0085 and 0.0181
between those of O. illyricus. The tree of individuals
(Fig. 8) emphasizes the differences between species
and suggests that the genetic distances among individuals are higher in O. taurus (longer branches) than
in O. illyricus (shorter branches). IUTS unambiguously clustered within the O. illyricus branch. AFLP
analysis did not provide any evidence of intraspecific
population geographical structure within each species.

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205

Figure 6. Phylogenetic tree resulting from Neighbour-joining reconstruction. Robustness was tested by bootstrapping
with 1000 replications. Only bootstrap values higher than 70% are shown in the figure. Five different haplotypes were
found in Onthophagus taurus (A1A5) and only one in Onthophagus illyricus. Names of the species are followed by the
code of the sequence in GenBank Database.

Table 3. Statistics of genetic differentiation among population in each species (A); statistics of genetic differentiation
among species in each site where the two species are found in syntopy (B); and statistics of genetic differentiation among
species in the whole sample (C)

A
B
C

Onthophagus taurus
Onthophagus illyricus
La Mandria
Brlog
Whole sample

HT

HW SE

HB SE

FST SE

0.243
0.196
0.410
0.441
0.417

0.236 0.012
0.182 0.012
0.197 0.027
0.221 0.028
0.206 0.022

0.007 0.000
0.014 0.000
0.213 0.000
0.22 0.000
0.211 0.000

0.027 0.051*
0.075 0.059**
0.517 0.065***
0.497 0.062***
0.504 0.052***

HT, total gene diversity; HW, average gene diversity within populations; HB, average gene diversity among populations in
excess of that observed within populations; FST, Wrights t fixation index for which significance was tested with 1000 random
permutations.
*P < 0.05; **P < 0.005; ***P < 0.001.

DISCUSSION
The present study investigated the morphological and
genetic differentiation pattern between two sympatric

dung beetle sister species. The effectiveness of both

morphometric and molecular analytical approaches
were compared to examine the nature of interspecific
relationships, to seek evidence of hybridization, and to

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206

A. PIZZO ET AL.

Figure 7. Scatterplot of the two first principal coordinate

scores obtained analyzing the dissimilarity matrix from
amplified fragment-length polymorphism data. The first
axis explains the 69.2% of the variation, whereas the second axis only the 5.53%. Black symbols, Onthophagus taurus; white symbols, Onthophagus illyricus; circles indicate
individuals from La Mandria (Italy), squares indicate individuals from Brlog (Croatia). IUTS are represented by
empty circles.

outline hypotheses of evolutionary and geographical

processes that might have led to interspecific differentiation and to the present-day partial sympatric and
syntopic distribution pattern.
Geometric morphometrics mostly failed to discriminate O. taurus from O. illyricus when external morphological characters were taken into account. Only
female head shape permitted a partial interspecific
discrimination. A similar pattern of overall morphological similarity was also observed in other animal
and plant sister species (Nakane, 1955; Yamazaki,
Goto & Nishida, 1997; Arlettaz, 1999; Hardig et al.,
2000; Dobigny, Baylac & Denys, 2002; Mathews et al.,
2002; Kawano, 2003). Animal genitalia often show a
distinct developmental relationship with other body
parts (Eberhard, 1990; Eberhard et al., 1998) and
sometimes exhibit unusual patterns of evolution
(Eberhard, 1985; Shapiro & Porter, 1989). It commonly occurs that the genitalia size of sympatric species of the same genus tends to differentiate, which
prevents overlapping, even when the two species
are very similar in their external morphology and

Figure 8. Tree of individuals of the two species from two

localities built using the matrix of the pairwise genetic
distances between each pair of individuals in the overall
amplified fragment-length polymorphism data set. Black
symbols, Onthophagus taurus; white symbols, Onthophagus illyricus; circles indicate individuals from La Mandria
(Italy), squares indicate individuals from Brlog (Croatia).
IUTS are represented by empty circles.

body size (Nakane, 1955; Butlin, 1987; Sirot, 2003;

Kawano, 2004). When two populations meet after a
period of divergence in allopatry, they may still have
structures similar enough to permit interpopulation
mating and to produce hybrids. In these cases, selection will tend to produce high divergence to reduce
wastage of reproductive effort (reproductive character
displacement) and to reduce gene flow by increasing
assortative mating (reinforcement) (Butlin, 1987;
Noor, 1999). Kawano (2002, 2003) demonstrated that
the genital morphology of beetle males of the genus
Chalcosoma and Odontolabis can be highly sensitive
to interspecific interactions, and that even small variations in the length of the penis appear to be suffi-

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GENETIC AND MORPHOLOGICAL DIFFERENTIATION IN DUNG BEETLE SISTER SPECIES

cient to prevent hybridation between two sympatric
species. Interspecific differentiation in the reproductive system morphology of O. taurus and O. illyricus
was higher, even if moderate, than in external morphology. This pattern is very similar to that found by
Kawano (2002, 2003, 2004) in many beetle species,
where sympatric character displacement was greater
in genitalia than in external body traits between
closely-related species with similar morphology and
behaviour. Kawano suggested that this displacement
might be more related to reproductive isolation than
to ecological niche divergence. Onthophagus taurus
and O. illyricus display a very similar behaviour and
share the same ecological niche: they feed, couple, and
nest on dung of herbivorous and omnivorous mammals and, when living in syntopy, individuals of both
species are also found in the same dung pads. However, the overlap of the reproductive activity period is
not complete. First, adults of O. taurus emerge in mid
spring, they immediately start nidification and egg
deposition, and young imagines appear at the beginning of summer (Lumaret & Kirk, 1991). Eggs
deposition of O. illyricus starts later, in June, and
imagines appear in July and August (Lumaret, 1990).
It might be assumed that interspecific competition
has led to an ecological temporal shift in the reproductive activity of the two species. It has been suggested
that an ecological character displacement, depending
on the presence of a competing species, also can
enhance the prezigotic isolation mechanism (Coyne &
Allen Orr, 2004). Moreover, genitalia may differentiate more readily because their variability is independent of the conservative ontogenetic network that
constrains the genetic variability of the other characters (Kawano, 2004). It is hypothesized that even the
moderate level of interspecific differentiation showed
by O. taurus and O. illyricus genitalia can be sufficient to avoid hybridization.
Usually, hybrids present morphological features
intermediate to parental species (Flner & Kraus,
1986; Gieler, Mader & Schwenk, 1999). In some
cases, they may resemble one of the parental species,
due to back-crossing or non-additive inheritance
(Gieler et al., 1999); in others, they may share alternatively some characters with each of the parental
species (Nice & Shapiro, 1999; Hardig et al., 2000;
Debussche & Thompson, 2002). In the species in the
present study, geometric morphometric analyses of
female head and genitalia of both sexes showed that
IUTS are included in the O. illyricus group, despite
their intermediate pubescence state of the elytra.
However, a morphological approach may not be sufficient to investigate hybridization events. Interspecific
hybridization among closely-related species can be
detected by genetic analyses because introgression
often results in merging of gene pools and incongruent

207

phylogenies (Arnold, 1997). Moreover, Neigel & Avise

(1986), when modelling lineage sorting during speciation, found that recently diverged sister species may
be paraphyletic with respect to mtDNA lineages for
some time after speciation. The reciprocal monophyly
of O. taurus and O. illyricus mtDNA lineages, with
IUTS sharing the species-specific haplotype of
O. illyricus populations, suggests that no introgression occurred after speciation. The nuclear markers
also showed an excellent interspecific discriminatory
power and IUTS, again, had the typical genetic profile
of the O. illyricus specimens and clustered within the
O. illyricus branch in the tree of individuals. This
pattern clearly indicates that the two species are
genetically and reproductively isolated. The genetic
diversity estimated by the Fst, as well as the Neis
interspecific genetic distances, also indicated a strong
differentiation between the two species. All results
were incongruent with the differentiation pattern
observed in recently diverged species or in species for
which hybridization events are known (Nice &
Shapiro, 1999). By contrast, evidence for divergence
between mtDNA sequences reflects long-term restriction of gene flow between lineages, whereas congruence of slow and fast genetic markers provides
evidence for historical isolation that persists today
(Crandall et al., 2000).
Genetic markers and morphological traits may differ in their evolutionary histories and mutation rates,
resulting in different patterns of evolution (Gorman &
Kim, 1976). Genetic changes may be substantial during morphological stasis (Strumbauer & Meyer, 1992)
and only if phenotypes respond to selection might they
experience strong directional selection (Gieler et al.,
1999). In the species pair O. taurusO. illyricus, a
clear genetic divergence corresponds to a moderate
genital differentiation and a very subtle morphological
divergence in external traits. These results indicate
that, in these dung beetles, genetic and morphological
evolution did not proceed in synchrony. The differentiation pattern outlined in such species clearly contrasts with that found in recently diverged Lycaeides
species (Nice & Shapiro, 1999), whose very small
interspecific distances (i.e. within the range commonly
observed for conspecific populations of other butterflies) are in congruence with minimal morphological
differentiation and with the existence of phenotypically intermediate individuals.
The absence of reliable evidence of hybridization
events (Stevens & Hogg, 2004) and historical or recent
ecological differentiation suggest that speciation
occurred in allopatry. Thus, it is plausible that the
present sympatric distribution of O. taurus and
O. illyricus populations is due to re-colonization or
range expansion events occurring when the reproductive isolation was fully accomplished.

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A. PIZZO ET AL.

The degree of genetic divergence between these sister species allowed us to estimate that they originated
in a time frame of 34 Mya, in the Pliocene, corresponding to an important frequency peak of speciation
for several beetle taxa (Davis, Scholtz & Philips, 2002;
Carisio et al., 2004; Ribera & Vogler, 2004). The
Pliocene climate changes could have fragmented the
distribution of the ancestral species in small isolated
populations, promoting speciation. Palaeoclimatic
fluctuations of the Plio-Pleistocene period are thought
to have heavily influenced the distribution of intraspecific genetic variation in numerous palearctic plants
and animals (Hewitt, 1996, 2000; Taberlet et al.,
1998). Many cycles of contraction/expansion of geographical ranges according to Pleistocene climatic
oscillations have led to the present genetic structure of
populations, species, and communities (Hewitt, 2000).
A population geographical structure is frequently
found in beetles as a result of extinctions, recolonization, and migration events during and after
the Pleistocene cold period (Reiss, Schwert & Ashworth, 1999; Carisio et al., 2004; Ribera & Vogler,
2004). Unexpectedly, the distribution of the genetic
variability observed in both O. taurus or O. illyricus
did not show appreciable geographical structure. Dispersal ability could have determined this unusual
absence of geographical population structure but
other factors, such as regional patterns of extinction or
recolonization, might have had important role
(Palumbi, 1995; Vogler, 1998). An elevated gene flow
among populations could be plausible for O. taurus,
which is known to be an extremely eurytopic species.
By contrast, O. illyricus is known as a more oligotopic
species (Lumaret, 1990; Borghesio, Palestrini & Passerin dEntreves, 2001; G. Dellacasa, pers. comm.) and
populations showed a unique mtDNA haplotype and
very similar AFLP profiles. In this case, dispersal ability and gene flow cannot exhaustively explain the
absence of geographical structure. However, a similar
marked interspecific and low intraspecific differentiation is described for two rodent sister species of the
genus Peromyscus (Zheng, Arbogast & Kenagy, 2003).
It has been suggested that this kind of pattern can
result from a recent expansion in population size and
geographical range. Nevertheless, further analyses
using other molecular markers must be conducted to
confirm this lack of geographical structure.

REFERENCES
Adams DC, Slice DE, Rohlf FJ. 2004. Geometric morphometrics: Ten years of progress following the revolution.
Italian Journal of Zoology 71: 516.
Albertson RC, Markert JA, Danley PD, Kocher TD. 1999.
Phylogeny of a rapidly evolving clade: the cichlid fishes of
Lake Malawi, East Africa. Proceedings of the National Acad-

emy of Sciences of the United States of America 92: 5107

5110.
Arlettaz R. 1999. Habitat selection as a major resource partitioning mechanism between the two sympatric sibling bat
species Myotis myotis and Myotis blythii. Journal of Animal
Ecology 68: 460.
Arnold ML. 1997. Natural hybridization and evolution. New
York, NY: Oxford University Press.
Arnqvist G, Edvardsson M, Friberg U, Nilsson T. 2000.
Sexual conflict promotes speciation in insects. Proceedings of
the National Academy of Sciences of the United States of
America 97: 1046010464.
Balthasar V. 1963. Monographie der Scarabaeidae und
Aphodiidae derpalaearktischen und orientalischen Region
(Coleoptera: Lamellicornia). Band 2, Coprinae. Prag: Verlag
der tschechoslowakischen Akademie der Wissenschaften.
Baraud J. 1992. Coloptres Scarabaeoidea dEurope. Paris:
Fderation Franaise des Socits des Sciences Naturelles.
Barluenga M, Meyer A. 2004. The Midas cichlid species complex: incipient sympatric speciation in Nicaraguan cichlid
fishes? Molecular Ecology 13: 20612076.
Barraclough TG, Vogler AP. 2000. Detecting the geographical pattern of speciation from the species-level phylogenies.
American Naturalist 155: 19434.
Bernatchez L, Dodson JJ. 1990. Allopatric origin of sympatric populations of lake Whitefish (Coregonus Clupeaformis)
as revealed by Mitochondrial DNA restriction analysis.
Evolution 44: 12631271.
Bookstein FL. 1989. Principal warps: Thin-plate splines and
the decomposition of deformations. I.E.E.E. Transactions on
Pattern Analysis and Machine Intelligence 11: 567585.
Bookstein FL. 1991. Morphometric tools for landmark data:
geometry and biology. Cambridge: Cambridge University
Press.
Borghesio L, Palestrini C, Passerin dEntreves P. 2001.
The dung beetle of the Gran Paradiso National Park: a preliminary analysis (Insecta: Coleoptera: Scarabaeoidea).
Journal of Mountain Ecology 6: 448.
Brower AVZ. 1994. Rapid morphological radiation and convergence among race of the butterfly Heliconius erato,
inferred from patterns of mitochondrial DNA evolution. Proceedings of the National Academy of Sciences of the United
States of America 91: 64916495.
Butlin R. 1987. Speciation by reinforcement. Trends in Ecology and Evolution 2: 813.
Carisio L, Cervella P, Palestrini C, Delpero M, Rolando
A. 2004. Patterns of genetic differentiation in three species
of the genus Trypocopris (Coleptera, Geotrupidae) inferred
from mtDNA and AFLP analyses. Journal of Biogeography
31: 11491162.
Congiu L, Dupanloup I, Paternello T, Fontana F, Rossi R,
Arlati G, Zane L. 2001. Identification of interspecific
hybrids by amplified fragment length polymorphism: the
case of sturgeon. Molecular Ecology 10: 23552359.
Coyne JA, Allen Orr H. 2004. Speciation. Sunderland, MA:
Sinauer Associates.
Crandall KA, Bininda-Emonds OR, Mace GM, Wayne
RK. 2000. Considering evolutionary processes in conser-

2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211

GENETIC AND MORPHOLOGICAL DIFFERENTIATION IN DUNG BEETLE SISTER SPECIES

vation biology. Trends in Ecology and Evolution 15: 290
295.
Darwin C. 1859. On the origin of the species by means of natural selection. London: Murray.
Davis ALV, Scholtz CH, Philips TK. 2002. Historical biogeography of scarabaeinae dung beetles. Journal of Biogeography 29: 12171256.
Dawson MN, Louie KD, Barlow M, Jacobs DK, Swift CC.
2002. Comparative phylogeography of sympatric sister species Clevelandia ios and Eucyclogobius newberryi (Teleostei,
Gobiidae), across the Califormia Transition Zone. Molecular
Ecology 11: 10651075.
De Knijff P, Denkers F, van Swelm DN, Kuiper M. 2001.
Genetic affinities within the herring gull Larus argentatus
assemblage revealed by AFLP genotyping. Journal of Molecular Evolution 52: 8593.
Debussche M, Thompson JD. 2002. Morphological differentiation among closely related species with disjunct distributions: a case study of Mediterranean Cyclamen L. subgen.
Psilanthum Schwars (Primulaceae). Botanical Journal of the
Linnean Society 139: 133144.
Dobigny G, Baylac M, Denys C. 2002. Geometric morphometrics, neural netwoks and diagnosis of sibling Taterillus
species (Rodentia, Gerbillinae). Biological Journal of the Linnean Society 77: 319327.
Eberhard WG. 1985. Sexual selection and animal genitalia.
Cambridge, MA: Harvard University Press.
Eberhard WG. 1990. Animal genitalia and female choice.
American Scientist 78: 134141.
Eberhard W, Huber BA, Rodriguez RL, Briceno RD,
Salas I, Rodriguez V. 1998. One size fits all? Relationships
between the size and degree of variation in genitalia and
other body parts in twenty species of insects and spiders.
Evolution 52: 415431.
Emlen DJ, Nijhout HF. 1999. Hormonal control of male horn
length dimorphism in the dung beetle Onthophagus taurus
(Coleoptera: Scarabaeidae). Journal of Insect Physiology 45:
4553.
Felsenstein J. 1985. Confidence limits on phylogenies: an
approach using the bootstrap. Evolution 39: 783791.
Felsenstein J. 1993. PHYLIP (Phylogeny Inference Package),
version 3.57c. Available at http://evolution.genetics.
washington.edu/phylip.html.
Flbner D, Kraus K. 1986. On the taxonomy of the Daphnia
hyalina-galeata complex (Crustacea: Cladocera). Hydrobiologia 137: 87115.
Gavrilets S, Waxman D. 2002. Sympatric speciation by sexual conflict. Proceedings of the National Academy of Sciences
of the United States of America 99: 1053310538.
Giannasi N, Thorpe RS, Malhotra A. 2001. The use of
Amplified Fragment length polymorphism in determining
species trees at fine taxonomic levels: analysis of a medically
important snake, Trimeresurus albolabris. Molecular Ecology 10: 419426.
Giebler S, Mader E, Schwenk K. 1999. Morphological
evolution and genetic differentiation in Daphnia species complexes. Journal of Evolutionary Biology 12:
710723.

209

Gitelman I, Davis CA. 1997. A novel DNA molecular weight

ladder. Trends in Genetics 770: 112.
Gorman GC, Kim YJ. 1976. Anolis lizard of the eastern Caribbean: a case study in evolution. II. Genetic relationships
and genetic variation of the bimaculatus group. Systematic
Zoology 25: 6277.
Hardig TM, Brunsfeld SJ, Fritz RS, Morgan M, Orians
CM. 2000. Morphological and molecular evidence for hybridization and introgression in a willow (Salix) hybrid zone.
Molecular Ecology 9: 924.
Hewitt GM. 1996. Some genetic consequences of ice ages, and
their role in divergence and speciation. Biological Journal of
the Linnean Society 58: 247276.
Hewitt G. 2000. The genetic legacy of the Quaternary ice ages.
Nature 405: 907913.
Howden HF, Gill BD. 1993. Mesoamerican Onthophagus
Latreille in the discranius and mirabilis species groups
(Coleoptera: Scarabaeidae). Canadian Journal of Entomology 125: 10911114.
Hunt J, Simmons LW. 2000. Maternal and paternal effects
on offspring phenotype in the dung beetle Onthophagus taurus. Evolution 54: 936941.
Hunt J, Simmons LW. 2002. The genetic effect of maternal
care: direct and indirect genetic effects on phenotype in the
dung beetle Onthophagus taurus. Proceedings of the
National Academy of Sciences of the United States of America
99: 68286832.
Janssens A. 1960. Faune de Belgique Insectes Coloptres
Lamellicornes. Bruxelles: Institut Royal des Sciences
Naturelles de Belgique, 411.
Kawano K. 2002. Character displacement in giant rhinoceros
beetles. American Naturalist 159: 255271.
Kawano K. 2003. Character displacement in stag beetles
(coleoptera, Lucanidae). Annals of the Entomological Society
of America 93: 198207.
Kawano K. 2004. Development stability and adaptive
variability of male genitalia in sexually dimorphic beetles.
American Naturalist 163: 115.
Kondrashov AS, Yampolsky LY, Shabalina SA. 1998. On
the sympatric origin of species by means of natural selection.
In: Howard DJ, Berlocher SH, eds. Endless forms: species
and speciation. New York, NY: Oxford University Press, 90
98.
Kumar S, Tamura K, Jakobsen IB, Nei M. 2001. MEGA 2.1:
molecular evolutionary genetics analysis software. Tempe,
AZ: Arizona State University.
Laird PW, Zijderveld A, Linders K, Rudnicki MA, Jaenisch R, Berns A. 1991. Simplified mammalian DANN isolation procedure. Nucleic Acid Research 19: 4293.
Liu J, Li P, Kucuktas H, Nicholson A, Tan G, Zheng X,
Argue BJ, Yant R, Dunham RA. 1999. Development of
AFLP markers for genetic linkage mapping analysis using
channel catfish and blue catfish interspecific hybrids.
Transactions of the American Fisheries Society 128: 317
327.
Liu J, Li P, Nicholson A, Dunham RA. 1998. Inheritance
and usefullness of AFLP markers in channel catfish (Ictalurus punctatus) and blue catfish (I. furcatus) and their F1, F2

2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211

210

A. PIZZO ET AL.

and backcross hybrids. Molecular and General Genetic 258:

260268.
Lohse GA, Lucht WH. 1992. Die Kfer Mitteleuropas, vol. 13.
Krefeld: Goecke and Evers.
Lumaret JP. 1990. Atlas Des Coloptres Scarabeides
Laparosticti de France. Paris: Musum National dHistoire
Naturelle, Inventaires de Faune et Flore 1.
Lumaret JP, Kirk AA. 1991. South temperate dung beetle.
In: Hanski I, Camberfort Y, eds. Dung beetle ecology. Princeton, NJ: Princeton University Press, 97115.
Lynch M, Milligan BG. 1994. Analysis of population
genetic structure with RAPD markers. Molecular Ecology
3: 9199.
Marcus LF, Corti M, Loy A, Naylor GJP, Slice DE. 1996.
Advances in morphometrics. NATO ASI Series A, vol. 284.
New York, NY: Plenum.
Martin-Piera F, Lopez-Colon JI. 2000. Coleoptera, Scarabaeoidea I. In: Ramos MA et al., eds. Fauna Iberica, vol. 14.
Madrid: Museo National de Ciencas Naturales, CSIC.
Mathews LM, Schubart CD, Neigel JE, Felder DL. 2002.
Genetic, ecological and behavioral divergence between two
sibling snapping shrimp species (Crustacea: Decapoda:
Alpheus). Molecular Ecology 11: 14271437.
Mayr E. 1963. Animal species and evolution. Cambridge, MA:
Harvard University Press.
Moczek AP. 1996. Male dimorphism in the scarab beetle
Onthophagus taurus Schreber 1759 (Scarabaeidae, Onthophagini) (Diplomarbeit): evolution and plasticity in a variable
environment. Master dissertation, Julius-MaximiliansUniversity, Wurzburg.
Moczek AP. 1998. Horn polyphenism in the beetle Onthophagus taurus: larval diet quality and plasticity in parental
investment determine adult body size and male horn morphology. Behavioral Ecology 9: 636641.
Moczek AP, Emlen DJ. 1999. Proximate determination of
male horn dimorphism in the beetle Onthophagus taurus
(Coleoptera: Scarabaeidae). Journal of Evolutionary Biology
12: 2737.
Moczek AP, Emlen DJ. 2000. Male horn dimorphism in the
scarab beetle Onthophagus taurus: do alternative reproductive tactics favour alternative phenotypes? Animal Behaviour 59: 459466.
Nakane T. 1955. Coloured illustrations of the insect of Japan:
Coleoptera. Osaka: Hoikusha.
Nei M. 1987. Molecular evolution genetics. New York, NY:
Columbia University Press.
Neigel JE, Avise JC. 1986. Phylogenetic relationships of
mitochondrial DNA under various demographic models of
speciation. In: Karlin S, Nevo E, eds. Evolutionary processes
and theory. New York, NY: Academic Press, 515534.
Nice CC, Shapiro AM. 1999. Molecular and morphological
divergence in the butterfly genus Lycaeides (Lepidoptera:
Lycaenidae) in North America: evidence of recent speciation.
Journal of Evolutionary Biology 12: 936950.
Noor MAF. 1999. Reinforcement and other consequences of
sympatry. Heredity 83: 503508.
Ogden R, Thorpe RS. 2002. The usefulness of amplified fragment lenght polymorphism markers for taxon discrimination

across graduated fine evolutionary levels in Caribbean

Anolis lizards. Molecular Ecology 11: 437445.
Palestrini C, Rolando A, Laiolo P. 2000. Allometric relationships and character evolution in Onthophagus taurus
(Coleoptera: Scarabaeidae). Canadian Journal of Zoology 78:
11991206.
Palumbi SR. 1995. Using genetics as an indirect estimator of
larval dispersal. In: McEdward LR, ed. Ecology of marine
invertebrate larvae. Boca Raton, FL: CRC Press, 369387.
Parsons YM, Shaw KL. 2001. Species boundaries and genetic
diversity among Hawaiian crickets of the genus Laupala
identified using amplified fragment length polymorphism.
Molecular Ecology 10: 17651772.
Paulian R, Baraud J. 1982. Faune des Coloptres de
France. Lucanoidea et Scarabaeoidea, vol. II. Paris: Editions
Lechevalier SARL, 477.
Reiss RA, Schwert DP, Ashworth AC. 1999. Molecular
genetic evidence of post-Pleistocene population divergence of
the arctic/alpine ground beetle Amara alpina (paykull)
(Coleoptera: Carabidae). Journal of Biogeography 26: 785
794.
Ribera I, Vogler AP. 2004. Speciation of Iberian diving
beetles in Pleistocene refugia (Coleoptera, Dytiscidae).
Molecular Ecology 13: 179193.
Rohlf FJ. 1990. Rotational fit (procrustes) methods. In: Rohlf
FJ, Bookstein FL, eds. Proceedings of the Michigan morphometrics workshop. Ann Arbor, MI: University of Michigan,
Museum of Zoology, 227236.
Rohlf FJ. 2003a. TpsDig 1.37. Available at http://life.bio.
sunysb.edu/morph/.
Rohlf FJ. 2003b. TpsRelw 1.33. Available at http://
life.bio.sunysb.edu/morph/.
Rohlf FJ. 19982002. NTSYS-PC Numerical Taxonomy and
Multivariate Analysis System V2 11. Setauket, NY: Exeter
Software.
Rohlf FJ, Marcus LF. 1993. A revolution in morphometrics.
Trends in Ecology and Evolution 8: 129132.
Rohlf FJ, Slice D. 1990. Extension of the procrustes method
for the optimal superimposition of landmarks. Systematic
Zoology 39: 4059.
Rosenberg MS. 2001. Fiddler crab claw shape variation: a
geometric morphometric analysis across the genus Uca
(Crustacea: Brachiura: Ocypodidae). Biological Journal of
the Linnean Society 75: 147162.
Saitou N, Nei M. 1987. The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Molecular
Biology and Evolution 4: 406425.
Salewski V. 2003. Satellite species in lampreys: a worldwide
trend for ecological speciation in sympatry? Journal of Fish
Biology 63: 267279.
Schluter D. 2000. The ecology of adaptive radiation. New
York, NY: Oxford University Press.
Seehausen O, Witte F, van Alphen JJM. 1998. Direct mate
choice maintains diversity in among sympatric cichlids in
Lake Victoria. Journal of Fish Biology 53: 3755.
Shapiro AM, Porter AH. 1989. The lock-and-key hypothesis:
Evolutionary and biosystematic interpretation of insect genitalia. Annals of Review of Entomology 34: 231245.

2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211

GENETIC AND MORPHOLOGICAL DIFFERENTIATION IN DUNG BEETLE SISTER SPECIES

Simmons LW, Tomkins JL, Hunt J. 1999. Sperm competition games played by dimorphic male beetles. Proceeding of
the Royal Society of London Series B 266: 145150.
Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook
P. 1994. Evolution, weighting and phylogenetic utility of
mitochondrial gene sequence and a compilation of conserved
polymerase chain reaction primers. Annals of the Entomological Society of America 87: 651701.
Sirot LK. 2003. The Evolution of insect mating structures
through sexual selection. Florida Entomologist 86: 124133.
Sneath PHA, Sokal RR. 1973. Numerical taxonomy. San
Francisco, CA: Freeman.
Stevens MI, Hogg ID. 2004. Population genetic structure of
New Zealands endemic corophiid amphipods: evidence of
allopatric speciation. Biological Journal of the Linnean Society 81: 119133.
Strumbauer C, Meyer A. 1992. Genetic divergence, speciation and morphological stasis in a lineage of African cichlid
fishes. Nature 358: 578581.
Swofford DL. 1990. PAUP: phylogenetic analysis using parsimony, version 4.0, Beta 10. Champaign, IL: Illinois Natural History Survey.
Taberlet P, Fumagalli L, Wust-Saucy AG, Cosson JF.
1998. Comparative phylogeography and postglacial colonization routes in Europe. Molecular Ecology 7: 453464.
Vekemans X. 2002. AFLP-SURVEY, version 1.0. Distributed
by the author. Bruxelles: Laboratoire de Gntique et Ecologie Vgtale, Universit Libre de Bruxelles.
Villalba S, Lobo MJ, Bleeker M, Martin-Piera F, Zardoya
R. 2002. Phylogenetic relationships of Iberian dung beetles
(Coleoptera: Scarabaeinae): insights on the evolution of nesting. Journal of Molecular Evolution 55: 116126.

211

Vogler AP. 1998. Extinction and the evolutionary process in

endangered species: what to conserve?. In: De Salle R, Schierwater B, eds. Molecular approaches to ecology and evolution. Basel: Birkhauser-Verlag, 191210.
Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T,
Hornes M, Frijters A, Pot J, Peleman J, Kuiper M,
Zabeu M. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acid Research 23: 44074414.
Wilkinson L. 1998. SYSTAT 8.0 Statistics. Chicago IL: SPSS
Inc.
Wright S. 1969. Evolution and the genetics of populations, vol.
2 The Theory of Gene Frequencies. Chicago, IL: University of
Chicago Press.
Yamazaki Y, Goto A, Nishida M. 1997. Mitochondrial DNA
sequence divergence between two cryptic species of
Lethenteron, with reference to an improved identification
technique. Journal of Fish Biology 62: 591609.
Zheng X, Arbogast BS, Kenagy GJ. 2003. Historical demography and genetic structure of sister species: deermice
(Peromyscus) in the North American temperate rain forest.
Molecular Ecology 12: 711724.
Zhivotovsky LA. 1999. Estimating population structure in
diploids with multilocus dominant DNA markers. Molecular
Ecology 8: 907913.
Zunino M. 1971. Importanza dellapparato genitale femminile
nella sistematica del genere Onthophagus Latr. Bollettino
Della Societ Entomologica Italiana 103: 2631.
Zunino M. 1979. Gruppi artificiali e gruppi naturali
negli Onthophagus (Coleoptera, Scarabaeoidea). Bollettino del Museo di Zoologia dellUniversit di Torino 1:
118.

2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211